Several authors have articulated the possibility of using the endogenous Pig-A gene as a reporter of somatic mutation (Araten et al., “Clonal Populations of Hematopoietic Cells with Paroxysmal Nocturnal Hemoglobinuria Genotype and Phenotype are Present in Normal Individuals,” Proc Natl Acad Sci USA 96:5209-5214 (1999); Chen et al., “Glycophosphatidylinositol-anchored Protein Deficiency as a Marker of Mutator Phenotypes in Cancer,” Cancer Res. 61:654-658 (2001)). As with the HPRT locus, Pig-A is located on the X-chromosome. Iida and colleagues isolated the human genomic gene, and found that it contains six exons over its 17 kb length (Iida et al., “Characterization of Genomic PIG-A Gene: A Gene for GPI Anchor Biosynthesis and Paroxysmal Nocturnal Hemoglobinuria,” Blood 83:3126-3131 (1994)). As demonstrated by Kawagoe et al., “Molecular cloning of Murine pig-a, a Gene for GPI-anchor Biosynthesis, and Demonstration of Interspecies Conservation of its Structure, Function, and Genetic Locus,” Genomics 23:566-574 (1994), there is a high degree of interspecies conservation of the gene's structure, function, and locus. The Pig-A gene product acts in the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis, and the entire process is thought to require at least 12 genes. Mutation of any one of these could theoretically result in GPI anchor deficiency. However, all other genes involved in GPI anchor synthesis are autosomal. Mutations on both alleles would have to occur to ablate expression of GPI anchors, and this is expected to be a very rare event. Thus, an inability to anchor GPI-linked proteins in the outer membrane is believed to be virtually equivalent to Pig-A mutation.
This key assumption, as well as practical aspects of assay development, greatly benefit from research on Paroxysmal Nocturnal Hemoglobinuria (PNH). PNH is a genetic disorder that affects 1 to 10 per million individuals, and is caused by a somatic Pig-A gene mutation within a bone marrow stem cell (Norris et al., “The Defect in Glycosylphosphatidylinositol Anchor Synthesis in Paroxysmal Nocturnal Hemoglobinuria,” Blood 83:816-821 (1994)). Since bone marrow stem cells are the precursors for the entire hematopoietic system, the gene mutation tends to affect numerous lineages. Erythrocytes, granulocytes and monocytes are typically affected. In a minority of cases, however, lymphocytes are also affected. A key finding is that all PNH clones to date exhibit mutation at the Pig-A locus (Nishimura et al., “Paroxysmal Nocturnal Hemoglobinuria: An Acquired Genetic Disease,” Am J Hematol 62:175-182 (1999)). Furthermore, an analysis of 146 PNH patients by Nishimura and colleagues provides important examples of the types of mutations that lead to GPI anchor deficiency. Single-base substitutions and frame-shift events are the most highly represented classes of mutation observed. Even so, there are three examples of large deletions (entire gene, 4 kb, and 737 base pairs), as well as a large insertion (88 base pairs). The mutations are widely distributed in the coding regions and splice sites, although others have found a somewhat higher frequency of missense mutations in exon 2 relative to other exons (Nafa et al., “The Spectrum of Somatic Mutations in the PIG-A Gene in Paroxysmal Nocturnal Hemoglobinuria Includes Large Deletions and Small Duplications,” Blood Cells Mol Dis 24:370-384 (1998)). Taken together, the PNH literature provides strong evidence that an in vivo assay based on the Pig-A gene would be sensitive to each important class of mutation.
In a report by Miura et al., “Development of an In Vivo Gene Mutation Assay Using the Endogenous Pig-A Gene: I. Flow Cytometric Detection of CD59-Negative Peripheral Red Blood Cells and CD48-Negative Spleen T-Cells From the Rat,” Environ. Molec. Mutagen. 49:614-621 (2008), a method for quantifying the frequency of mutant phenotype erythrocytes was identified. In that flow cytometry-based assay, anti-CD45 antibody was used to differentiate leukocytes from erythrocytes, and anti-CD59-FITC was used to distinguish mutant phenotype erythrocytes from wild-type erythrocytes. The authors also described a second approach whereby the fluorescent reagent FLAER and flow cytometry could be used to quantify the frequency of mutant phenotype erythrocytes. However, these approaches did not differentiate mature erythrocytes from the immature fraction of erythrocytes (reticulocytes). This is a significant disadvantage of the approach of Miura et al., because differential staining of mature and immature erythrocytes allows one to determine the percentage of reticulocytes among total erythrocytes simultaneously with Pig-A mutation measurements. These percent reticulocyte values provide important information regarding bone marrow toxicity, a parameter that is valuable for interpreting any genotoxicity endpoint that is based on hematopoietic cells. Differentially staining reticulocytes and mature erythrocytes also allows one to measure Pig-A mutation frequency in both the total RBC cohort as well as the reticulocyte fraction. The latter measurement is valuable for some experimental designs, since maximal mutagenic responses are obtained in this fraction of cells sooner than those observed in the total erythrocyte pool. Furthermore, the approach of Miura et al. for distinguishing erythrocytes from leukocytes was less than ideal. Namely, in their hands, anti-CD45 did not afford clear resolution of nucleated cells from erythrocytes. Rather than distinct populations, a continuum of CD45-associated fluorescent events was observed. The consequence of this is contamination of the erythrocyte analyses with leukocytes that failed to exhibit sufficient differential fluorescent resolution. This likely contributed to the high and variable baseline mutation frequencies that were reported by these investigators.
In U.S. Pat. No. 7,824,874 to Dertinger, a method of enumerating Pig-A mutant cell frequency from peripheral blood samples is identified. The described methodology uses a three-color labeling approach to distinguish GPI anchor-deficient cells from GPI anchor-expressing cells, platelets from other blood cells, and reticulocytes from erythrocytes. In U.S. Patent Application Publ. No. US20090311706 to Dertinger and Phonethepswath et al., “Erythrocyte-based Pig-a Gene Mutation Assay: Demonstration of Cross-Species Potential,” Mutat. Res. 657:122-126 (2008), another method of enumerating Pig-A mutation frequency from peripheral blood samples is identified. In this method, the peripheral blood sample is treated in a manner to substantially separate RBCs from platelets and leukocytes, thereby enriching the sample for RBCs and making the method less susceptible to spuriously high readings. However, in both cases, the time required to evaluate millions of cells, especially reticulocytes, for the Pig-A mutant phenotype is a very time-consuming process, and most analyses are therefore based on suboptimal numbers of total cells interrogated for the mutant phenotype. This situation leads to less reliable estimates of mutation frequency, especially in situations when mutation frequency is low, as is typically the case in individuals that have not been exposed to potent mutagen(s). It would be desirable to obtain an assay that can identify and quantify many more Pig-A mutant phenotype cells per unit time. Such an assay would be more practical to perform, in terms of efficiently studying large numbers of specimens, and it would be endowed with greater reliability and sensitivity, i.e., greater power to detect modest changes to the frequency of mutant phenotype erythrocytes and/or mutant phenotype reticulocytes.
The present invention is directed to overcoming these and other deficiencies in the prior art.